Identification of Signaling Pathways by Which CD40 Stimulates Autophagy and Antimicrobial Activity against Toxoplasma gondii in Macrophages

Abstract

CD40 is an important stimulator of autophagy and autophagic killing of Toxoplasma gondii in host cells. In contrast to autophagy induced by nutrient deprivation or pattern recognition receptors, less is known about the effects of cell-mediated immunity on Beclin 1 and ULK1, key regulators of autophagy. Here we studied the molecular mechanisms by which CD40 stimulates autophagy in macrophages. CD40 ligation caused biphasic Jun N-terminal protein kinase (JNK) phosphorylation. The second phase of JNK phosphorylation was dependent on autocrine production of tumor necrosis factor alpha (TNF-␣). TNF-␣ and JNK signaling were required for the CD40-induced increase in autophagy. JNK signaling downstream of CD40 caused Ser-87 phosphorylation of Bcl-2 and dissociation between Bcl-2 and Beclin 1, an event known to stimulate the autophagic function of Beclin 1. However, TNF-␣ alone was unable to stimulate autophagy. CD40 also stimulated autophagy via a pathway that included calcium/calmodulin-dependent kinase kinase ␤ (CaMKK␤), AMP-activated protein kinase (AMPK), and ULK1. CD40 caused AMPK phosphorylation at its activating site, Thr-172. This effect was mediated by CaMKK␤ and was not impaired by neutralization of TNF-␣. CD40 triggered AMPK-dependent Ser-555 phosphorylation of ULK1. CaMKK␤, AMPK, and ULK1 were required for CD40-induced increase in autophagy. CD40-mediated autophagic killing of Toxoplasma gondii is known to require TNF-␣. Knockdown of JNK, CaMKK␤, AMPK, or ULK1 prevented T. gondii killing in CD40-activated macrophages. The second phase of JNK phosphorylation-Bcl-2 phosphorylation-Bcl-2-Beclin 1 dissociation and AMPK phosphorylation-ULK1 phosphorylation occurred simultaneously at ϳ4 h post-CD40 stimulation. Thus, CaMKK␤ and TNF-␣ are upstream molecules by which CD40 acts on ULK1 and Beclin 1 to stimulate autophagy and killing of T. gondii.

Apicomplexan autophagy and modulation of autophagy in parasite-infected host cells

“…CD40 [a member of the tumor necrosis factor receptor superfamily] signaling has also been shown to trigger autophagic elimination of T. gondii independently of IFN-γ and IRGs [33] , [34] , [35] , [36] . This anti-parasitic activity also depends on proteins of the autophagy machinery, and the sequestration and degradation of intracellular tachyzoites occurs possibly through classical autophagy, although this remains to be more firmly established.…”

Toxoplasma gondiiinduces prolonged host epidermal growth factor receptor signalling to prevent parasite elimination by autophagy: Perspectives for in vivo control of the parasite

“…CD40 ligation acts on ULK1, a central initiator of autophagy in mammalian cells (Chan, Longatti, McKnight, & Tooze, , Itakura & Mizushima, , Russell et al, ), causing its S555 phosphorylation (marker of ULK1 activation; Liu, Lopez Corcino, Portillo, Miao, & Subauste, ), induces dissociation of Beclin 1 from Bcl‐2 (Liu et al, ), a process that enables Beclin 1 to stimulate autophagosome formation (Pattingre et al, ), and activates PKR (Ogolla et al, ), a protein that stimulates autophagy (Talloczy, Virgin, & Levine, ). As a result, CD40 increases autophagosome formation and maturation to autolysosomes (autophagy flux; Ogolla et al, , Van Grol, Muniz‐Feliciano, Portillo, Bonilha, & Subauste, , Liu et al, ). CD40 ligation in haematopoietic and nonhaematopoietic cells from humans and mice leads to accumulation of the autophagy protein LC3 around the parasite followed by recruitment of the lysosomal marker LAMP‐1 and killing of T .…”

“…CD40 ligation in haematopoietic and nonhaematopoietic cells from humans and mice leads to accumulation of the autophagy protein LC3 around the parasite followed by recruitment of the lysosomal marker LAMP‐1 and killing of T . gondii that is dependent on ULK1 (the kinase that triggers autophagy) as well as PKR, the autophagy proteins Beclin 1, ATG5, ATG7, and lysosomal enzymes (Andrade, Wessendarp, Gubbels, Striepen, & Subauste, ; Liu et al, ; Ogolla et al, ; Portillo et al, ; Van Grol et al, ).…”

The role of host autophagy machinery in controllingToxoplasmainfection

“…Moreover, canonical autophagy seems involved, as CD40 signaling appears to act on autophagy. More precisely, it acts on LC3 recruitment around the parasites [65] through the stimulation of upstream regulators such as Beclin 1 [63], a regulatory subunit of autophagy-promoting PtdIns3K (Figure 2(a)), and ULK1 [66], a kinase that regulates this complex (Figure 2 (a)) [67]. In non-hematopoietic cells, the down-regulation of autophagy proteins (Beclin 1 or ATG7), or the use of the PtdIns3K-inhibitor 3-methyladenine to block autophagy, Figure 3.…”